The present invention-relates to a process for fractionating polyethylene glycol-protein adducts.
Polyethylene glycol is a long chain, linear synthetic polymer composed of ethylene oxide units, HO(CH2CH2O)nCH2CH2OH, in which n can vary to provide compounds with molecular weights from 200-20,000. It is non-toxic and has been administered orally and intravenously to humans (PEG-adenosine deaminase for severe combined immunodeficiency disease; PEG-asparaginase for acute lymphoblastic leukaemia; PEG-superoxide dimutase for oxygen toxicity (3-7)). PEG can be coupled to proteins following appropriate derivatisation of the OH groups of the PEG. The NH2 groups of lysine side chains are particularly accessible sites and either few or many sites can be modified. Given, adequate technology for their production, PEG-modified proteins have numerous therapeutic and other applications. Many proteins of potential clinical use have extremely short half lives necessitating administration by continuous infusion (an expensive, unpleasant and potentially hazardous procedure). PEG modification extends plasma half lives and has been used to increase the bio-availability of enzymes (see below). Reduction of antigenicity of proteins is also produced by PEG modification and this will extend their clinical use allowing more protracted administration. In addition, with proteins having pleiotropic biological effects, PEG modification creates products with a new spectrum of activities, because of differential loss of separate biological properties. With antibodies, for example, PEG modification dissociates antibody binding and complement fixing activities. PEG modification also alters biochemical and physical properties of proteins in ways that may increase their usefulness (e.g. increased solubility; increased resistance to proteolytic degradation; altered kinetics, pH and/or temperature optima and changed substrate specificity of enzymes) This covalent modification of proteins has a number of consequences:
(i) Increased Plasma Half-life:
This has been found with numerous proteins (See Table 1 and reference 8-17) and has already been exploited clinically. Two children with adenosine deaminase deficiency were successfully treated with PEG-modified bovine adenosine deaminase (18). In acute lymphoblastic leukaemia, 74% of 20 patients achieved complete or partial remissions with PEG-asparaginase (5). Increased half-life and enhanced antitumour potency was also observed with PEG-interleukin 2 in the Meth A murine sarcoma model (19). The basis for this increase in half-life is not understood and may include such factors as reduction of glomerular filtration of small peptides because of the increase in size due to PEG modification (19). The increase in biological potency (which may relate to other phenomena in addition to the increased half-life) is potentially very important in the use of PEG-cytokine adducts as pharmacological agents in cancer therapy.
ii) Altered Biochemical and Physical Properties:
These include increased solubility (20), because of the addition of hydrophilic PEG chains (useful for proteins like interleukin 2 which have limited solubility at physiological pH (19)), increased resistance to proteolytic degradation (21), changes in kinetics or pH and temperature optima or substrate specificity of enzymes (10,20,22,23)). Relevant to the present project are observations which suggest differential effect on function e.g. complement fixing activity and antigen-binding are lost and retained respectively after PEG-modification of IgG (24). PEG-ribonuclease has an altered activity for high but not low molecular weight substrates (25). To some extent, these effects can be controlled by varying the number of sites on the protein modified and the length of the PEG polymer.
(iii) Reduced Antigenicity:
This includes reduced ability to react to antibodies to the unmodified protein and low immunogenicity of the PEG-proteins themselves (26).
Coupling of PEG to proteins-is usually achieved by activation of the hydroxyl groups of PEG with a suitable reagent that can be fully substituted by nucleophilic groups in the protein (mainly lysine E-amino groups) (27). Cyanuric chloride has been the most widely used agent for activation of PEG and this requires a very basic pH for the subsequent coupling step with the protein to be modified (28,27). In order to avoid these adverse conditions (particularly important when dealing with labile proteins like growth factors), alternative methods have been sought. However, 1,1xe2x80x2-carbonyldiimidazole requires very long times for the coupling step (14) and using phenylchloroformates does not avoid the need for basic pH (25).
Although much of this information has been available for many years, PEG-proteins are not widely available commercially.
Tresyl chloride (2,2,2,-trifluoroethane-sulphonyl chloride) has proved useful for activating agarose and other solid supports carrying hydroxyl groups so that they may be coupled to proteins. The attraction of this method is that coupling to proteins takes place quickly and under very mild conditions (28,29). We have successfully applied this approach to the activation of monomethoxyPEG (MPEG), this has a single free derivatisable OH group. We have demonstrated the subsequent coupling of MPEG to both antibodies (30) and albumin (see example 1), under mild conditions (pH 7.5 phosphate buffer, at room temperature). An advantage over previous techniques is that the reaction mixture is innocuous and does not have to be removed before the PEG-protein is used. We have also developed a technique to neutralise excess tresyl-PEG after the coupling step (to prevent reaction with other proteins and/or cells) thus avoiding the need for laborious chromatography or ultrafiltration to remove it. These improvements are of importance when applying the method to labile growth factor proteins, which are notoriously sensitive to manipulations such as ultrafiltration.
Given acceptable (non-denaturing) conditions for the coupling step, there are two main variables that will affect the biological properties of the PEG-proteins and these may be controlled in the manufacturing process. One is the length of the PEG molecules attached per protein molecule and the second is the number of PEG molecules per protein.
Where proteins have several lysine groups, varying the molar ratio of activated MPEG to protein influences the degrees of substitution markedly (see example 2). What is needed is a means of determining what degree of substitution gives the best outcome vis a vis the desired biological properties and then to devise a manufacturing scheme which best achieves this degree of substitution. Biochemical monitoring methods are cumbersome (2) and do not give an estimate of the variability in substitution of the population of modified protein molecules. They also do not allow recovery of materials with different degrees of substitution (the latter is difficult to control by altering molar ratios, since a wide distribution of degrees of substitution is observed at any given molar ratio, until full substitution is approached at high molar ratios (see example 2). Both analytical work to determine which degree of substitution produces the optimum effect and the manufacturing process requires a means of fractionating peptides/proteins with different (and preferably precisely defined) degrees of substitution. The problem is likely to be widespread since most clinically useful proteins have several lysine residues (Table II).
Although PEG-modification of over a dozen proteins has now been described, frequently in extensive practical detail, little attention has been given to the PEG-proteins being heterogeneous in their degree of substitution with PEG (23).
The partitioning behaviour of PEG-protein adducts in PEG-containing aqueous biphasic systems has not been previously defined, nor has the relationship between degree of PEG substitution and partitioning coefficient. On investigating the partitioning behaviour in such systems we have surprisingly discovered that PEG-containing aqueous biphasic systems are uniquely tailored to separating PEG-proteins sensitively and can thus be used to monitor the effect of degree of modification on biological properties and, on a bulk scale to prepare PEG proteins 6f specified degrees of substitution.
The invention therefore provides a process for fractionating a mixture of PEG-protein adducts comprising partitioning the PEG-protein adducts in a PEG-containing aqueous biphasic system. Preferably the process further comprises the step of recovering a PEG-protein adduct of pre-determined degree of PEG substitution from one phase of the biphasic system. Whilst any PEG protein adduct mixture may be fractionated in accordance with the invention, it is preferred to use adducts of monomethoxyPEG preferably those formed by reaction of the protein with tresyl monomethoxyPEG, (TMPEG). In a particular aspect of the invention, unreacted TMPEG is destroyed or the adduct-forming reaction is quenched, by addition of lysine or albumin. Partitioning may be performed batchwise or continuously, for instance by counter currents of the two phases and may be repeated to obtain additional fractionation.